The invention relates to nucleic acid molecules encoding GABAB receptors, and to methods for screening for compounds that are inhibitors of transient lower esophageal sphincter relaxations (TLESR).
GABAB Receptors
GABA (4-aminobutanoic acid) is an endogenous neurotransmitter in the central and peripheral nervous systems. Receptors for GABA have traditionally been divided into GABAA and GABAB receptor subtypes. GABAB receptors (for a review see Kerr, D. I. B. and Ong, J. (1995) Pharmac. Ther. vol. 67, pp.187-246) belong to the superfamily of G-protein coupled receptors. GABAB receptor agonists are useful in the treatment of central nervous system (CNS) disorders, such as for inducing muscle relaxation in spinal spasticity, cardiovascular disorders, asthma, and gut motility disorders such as irritable bowel syndrome; and as prokinetic and anti-tussive agents. GABAB receptor agonists have also been disclosed as useful in the treatment of emesis (WO 96/11680).
The cloning of the rat GABAB receptors GABABR1a (SEQ ID NOs: 44 and 45) and GABABR1b (SEQ ID NOs: 46 and 47) was disclosed by Kaupmann et al. ((1997) Nature, vol. 386, 239-246). The mature rat GABABR1b differs from GABABR1a in that the N-terminal 147 residues are replaced by 18 different residues. It is thought that the rat GABABR1a and GABABR1b receptor variants are derived from the same gene by alternative splicing. Cloning of the human GABABR1b receptor was disclosed in WO97/46675.
Reflux
In some humans, the lower esophageal sphincter (LES) is prone to relaxing more frequently than in other humans. As a consequence, fluid from the stomach can pass into the esophagus because the mechanical barrier is temporarily lost at such times, an event hereinafter referred to as xe2x80x9creflux.xe2x80x9d
Gastro-esophageal reflux disease (GERD) is the most prevalent upper gastrointestinal tract disease. Conventional therapies have sought to reduce gastric acid secretion, or reduce esophageal acid exposure by enhancing esophageal clearance, lower esophageal sphincter tone, and gastric emptying. The major mechanism behind reflux has been considered to depend on a hypotonic lower esophageal sphincter. However, recent research (e.g., Holloway and Dent (1990) Gastroenterol. Clin. N. Amer. 19, 517-535) has shown that most reflux episodes occur during transient lower esophageal sphincter relaxations (TLESR), i.e., relaxations not triggered by swallowing. It has also been shown that gastric acid secretion usually is normal in patients with GERD.
The present invention provides nucleic acid molecules encoding human and canine GABAB receptors. These nucleic acid molecules make it possible to screen for compounds that are agonists or antagonists of GABAB receptors, e.g., to identify compounds which are inhibitors of TLESR.
Consequently, the invention provides an isolated nucleic acid molecule encoding a human or canine GABAB receptor, or a conservative variant thereof. An xe2x80x9cisolated nucleic acidxe2x80x9d is a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes. The term therefor covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Specifically excluded from this definition are nucleic acids present in mixtures of (i) DNA molecules, (ii) transfected cells, and (iii) cell clones: e.g., as these occur in a DNA library such as a cDNA or genomic DNA library.
In various embodiments, the nucleic acid molecule encodes a human GABAB receptor 1a (SEQ ID NOs: 48 and 49), 1b (SEQ ID NOs: 50 and 51), 1c (SEQ ID NOs: 54 and 55) or 1d (SEQ ID NOs: 56 and 57); or a canine GABAB receptor 1a (SEQ ID NOs: 52 and 53) or 1c (SEQ ID NOs: 58 and 59). Accordingly, the invention includes the following nucleic acid molecules:
(1) a nucleic acid molecule that includes a nucleotide sequence set forth as SEQ ID NO: 48, 50, 52, 54, 56, or 58, or a degenerate variant thereof;
(2) an RNA molecule that includes a nucleotide sequence set forth as SEQ ID NO: 48, 50, 52, 54, 56, or 58, or a degenerate variant thereof, wherein T is replaced by U;
(3) a nucleic acid molecule that includes a nucleotide sequence that is capable of hybridizing under stringent conditions (e.g., is complementary) to a nucleotide sequence of (1) or (2), or to the complement of (1) or (2); and
(4) nucleic acid fragments that are at least 15 base pairs in length and which hybridize under stringent conditions to genomic DNA encoding the human or canine GABAB polypeptides described herein, or to the complement of such genomic DNA.
The invention also includes isolated nucleic acid molecules corresponding to genomic sequences encoding human GABAB receptors (SEQ ID NOs: 60 and 61), as well as nucleic acid molecules (set forth as SEQ ID NO: 70, 72, 74, 76, 78, 80, 82, and 84) encoding additional isoforms of the human GABAB receptor, which isoforms are generated by alternative splicing.
The nucleic acid molecules of the invention are not limited strictly to molecules including the sequences set forth as SEQ ID NOs: 48, 50, 52, 54, 56 or 58. Rather, the invention encompasses nucleic acid molecules carrying modifications such as substitutions, small deletions, insertions, or inversions, which nevertheless encode proteins having substantially the biochemical activity of the GABAB receptors according to the invention, and/or which can serve as hybridization probes for identifying a nucleic acid with one of the disclosed sequences. Included in the invention are nucleic acid molecules, the nucleotide sequence of which is at least 95% identical (e.g., at least 96%, 97%, 98%, or 99% identical) to the nucleotide sequence shown as SEQ ID NO: 48, 50, 52, 54, 56, or 58 in the Sequence Listing.
The determination of percent identity or homology between two sequences is accomplished using the algorithm of Karlin and Altschul (1990) Proc. Nat""l Acad. Sci. USA 87: 2264-2268, modified as in Karlin and Altschul (1993) Proc. Nat""l Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches are performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used. See http://www.ncbi.nlm.nih.gov.
The term xe2x80x9cstringent hybridization conditionsxe2x80x9d is known in the art from standard protocols (e.g., Current Protocols in Molecular Biology, editors F. Ausubel et al., John Wiley and Sons, Inc. 1994) and is to be understood as conditions as stringent as those defined by the following: hybridization to filter-bound DNA in 0.5 M NaHPO4 (pH 7.2), 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at +65xc2x0 C., and washing in 0.1xc3x97SSC/0.1% SDS at +68xc2x0 C.
Also included in the invention is a nucleic acid molecule that has a nucleotide sequence which is a degenerate variant of a nucleic acid disclosed herein, e.g., SEQ ID NOs: 48, 50, 52, 54, 56, and 58. A sequential grouping of three nucleotides, a xe2x80x9ccodon,xe2x80x9d encodes one amino acid. Since there are 64 possible codons, but only 20 natural amino acids, most amino acids are encoded by more than one codon. This natural xe2x80x9cdegeneracyxe2x80x9d or xe2x80x9credundancyxe2x80x9d of the genetic code is well known in the art. It will thus be appreciated that the nucleic acid sequences shown in the Sequence Listing provide only an example within a large but definite group of nucleic acid sequences that will encode the polypeptides as described above.
The invention also includes an isolated polypeptide encoded by a nucleic acid of the invention. An xe2x80x9cisolatedxe2x80x9d polypeptide is a polypeptide that is substantially free from the proteins and other naturally occurring organic molecules with which it is naturally associated. Purity can be measured by any art-known method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC.
An isolated polypeptide may be obtained, for example, by extraction from a natural source (e.g., a human cell); by expression of a recombinant nucleic acid encoding the polypeptide; or by chemical synthesis of the polypeptide. In the context of a polypeptide obtained by extraction from a natural source, xe2x80x9csubstantially freexe2x80x9d means that the polypeptide constitutes at least 60% (e.g., at least 75%, 90%, or 99%) of the dry weight of the preparation. A protein that is chemically synthesized, or produced from a source different from the source from which the protein naturally originates, is by definition substantially free from its naturally associated components. Thus, an isolated polypeptide includes recombinant polypeptides synthesized, for example, in vivo, e.g., in the milk of transgenic animals, or in vitro, e.g., in a mammalian cell line, in E. coli or another single-celled microorganism, or in insect cells.
In various embodiments, the polypeptide of the invention has an amino acid sequence as set forth in SEQ ID NO: 49, 51, 53 55, 57, 59, 71, 73, 75, 77, 79, 81, 83, and 85. However, polypeptides of the present invention are not to limited to those having an amino acid sequence identical to one of SEQ ID NOs: 49, 51, 53, 55, 59, 71, 73, 75, 77, 79, 81, 83, or 85 in the Sequence Listing. Rather, the invention also encompasses conservative variants of the disclosed sequences. xe2x80x9cConservative variantsxe2x80x9d include substitutions within the following groups: glycine and alanine; valine, alanine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine, and threonine; lysine, arginine, and histidine; and phenylalanine and tyrosine.
Also included in the invention are polypeptides carrying modifications such as substitutions, small deletions, insertions, or inversions, which polypeptides nevertheless have substantially the biological activities of the GABAB receptor. Consequently, included in the invention is a polypeptide, the amino acid sequence of which is at least 95% identical (e.g., at least 96%, 97%, 98%, or 99% identical) to an amino acid sequence set forth as SEQ ID NO: 49, 51, 53, 55, 57 59, 71, 73, 75, 77, 79, 81, 83, or 85 in the Sequence Listing. xe2x80x9cPercent identityxe2x80x9d is defined in accordance with the algorithm described above.
Also included in the invention are polypeptides of the invention that have been post-translationally modified, e.g., by cleavage of an N-terminal signal sequence, which can be, e.g., 1 to 25 amino acids long.
The invention also includes a vector that contains a nucleic acid molecule of the present invention. The vector can, e.g., be a replicable expression vector that is capable of mediating the expression of a nucleic acid molecule of the invention. A xe2x80x9creplicablexe2x80x9d vector is able to replicate in a given type of host cell into which it has been introduced. Examples of suitable vectors include virus-based vectors (e.g., bacteriophages, retroviruses, adenoviruses, herpes viruses, polio viruses, and vaccinia viruses), cosmids, plasmids, and other recombination vectors. Nucleic acid molecules can be inserted into vectors by methods well known in the art.
Also included in the invention is a host cell harboring a nucleic acid (e.g., on a vector) of the invention. Without limitation, such a host cell can be a prokaryotic cell, a unicellular eukaryotic cell, or a cell derived from a multicellular organism. For example, the host cell can be a bacterial cell, such as an E. coli cell; a yeast cell, such as Saccharomyces cerevisiae or Pichia pastoris; an insect cell, an amphibian cell (e.g., a frog oocyte), or a mammalian cell. It is preferably not a neuron, e.g., a human, dog, rat or other mammalian neuron. Conventional methods can be employed to introduce the vector into the host cell.
Host cells containing nucleic acids of the invention can be used to produce a GABAB receptor polypeptide of the invention or a conservative variant thereof. Generally, the process includes culturing a host cell as defined above under conditions such that the polypeptide is produced, and recovering the polypeptide.
A further aspect of the invention is a method for determining whether a test compound is an inhibitor of TLESR. The method entails (a) expressing in a cell (preferably a cell that does not naturally express the GABAB receptor, such as a fibroblast or other non-neural cell) a nucleic acid molecule that includes a nucleotide sequence of the invention, thereby producing a cell having on its surface a GABAB receptor or a conservative variant thereof; (b) contacting the GABAB receptor or conservative variant with a test compound; and (c) detecting binding of the test compound to the GABAB receptor or conservative variant, wherein binding of the test compound to the GABAB receptor or conservative variant indicates that the test compound is an inhibitor of TLESR. This activity can be further validated by other in vitro or in vivo tests: e.g., by administration of the test compound to an animal model for this condition. It should be understood that this aspect of the invention is not limited to use of human and canine GABAB receptors, but rather encompasses the use of any GABAB receptor for screening for compounds which are inhibitors of TLESRs.
Nucleic acid molecules encoding human or canine GABAB receptors also can be used in a related method for screening for compounds that are agonists or antagonists. Generally, in this method, binding is detected by detecting activation, or inhibition of activation, of the GABAB receptor or a conservative variant thereof, wherein activation indicates that the test compound is an agonist of the GABAB receptor, and inhibition of activation indicates that the test compound is an antagonist of the GABAB receptor.
The screening methods according to the invention can e.g., comprise the steps (a) transforming a cultured cell with a nucleic acid molecule encoding a GABAB receptor, so that a GABAB receptor is expressed on the surface of the cell; (b) contacting a test compound with the cell; and (c) determining whether the test compound binds to, and/or activates, the GABAB receptor.
GABAB receptor-expressing cells, transgenic animals, or cells and tissues derived therefrom can be used to screen substance libraries (i.e., libraries of test compounds) for antagonist or agonist activity. For this purpose, GABAB receptor expression may be directed to cells and tissues containing, either naturally or artificially, the necessary components allowing correct receptor transport and processing as well as coupling to second messenger pathways. Screening may be performed as ligand binding assays or functional assays. For screening, cells and tissues can be prepared in various ways, each uniquely suited to its purpose. Ligand binding assays can be performed in vivo or in vitro using, e.g., radiolabelled GABA. Functional assays (e.g., Ca++-responses, cAMP-responses, and effects on K+ channels) can be performed in living cells, broken cells, isolated cell membranes, tissues, or living animals. To facilitate measurement of physiological GABAB receptor mediated responses, GABAB receptors may be co-expressed with promiscuous G-proteins, e.g., Gxcex116 or Gqi5, increasing G-protein coupling. Another way to increase G-protein coupling is to fuse the GABAB receptor to appropriate G-proteins using standard molecular techniques. To further improve readouts in Ca++-response assays, GABAB receptors can be co-expresses with aequorin, a photoprotein cloned from the luminescent jellyfish Aequorea victoria. 
The invention also provides a pharmaceutical composition that includes a GABAB receptor (e.g., a soluble receptor), or a conservative variant thereof, and at least one of (a) a pharmaceutically acceptable carrier and (b) a pharmaceutically acceptable diluent.
The pharmaceutical composition can be used in methods of treating conditions involving GABA-dysfunction, e.g., epilepsy, psychiatric disorders such as depression and anxiety, cognitive dysfunction, gastroesophageal reflux disease, emesis, irritable bowel syndrome, dyspepsia, spasticity, arthritis, allergies, autoimmune diseases, neoplastic diseases, pain, and infectious diseases. Typically, the GABAB receptor is a soluble form of the GABAB receptor, such as the human GABAB receptor 1c or 1d or a conservative variant thereof.
A soluble form of the receptor can be a form that lacks some or all of the membrane-spanning domains of the wild-type receptor protein, but retains the ligand-binding portion or portions of the receptor. The membrane-spanning domains are readily identified by their predominance of non-polar amino acid residues, and/or by comparison with related receptors (e.g., other G-protein receptors).
Soluble forms of the GABAB receptor can be produced by culturing a host cell containing a vector that includes a nucleic acid encoding the soluble GABAB receptor under conditions such that the GABAB receptor polypeptide is produced. The polypeptide then is recovered, and a pharmaceutical composition containing the polypeptide is administered to a mammal (e.g., a human or dog) in need thereof.
In a related aspect, the invention provides a method for diagnosing a mammal as having a condition involving altered levels of GABAB receptors in body fluid (e.g., serum or cerebrospinal fluid). Such conditions include epilepsy, psychiatric disorders, cognitive dysfunction, gastroesophageal reflux disease, emesis, irritable bowel syndrome, dyspepsia, spasticity, arthritis, allergies, auto immune diseases, neoplastic diseases, pain, and infectious diseases. Diagnosis involves measuring the level of GABAB receptor in a body fluid of a mammal (e.g., a human), wherein an increase or decrease in the level of GABAB receptor, relative to the level found in a normal mammal, indicates that the mammal has a condition involving altered levels of GABAB receptors in body fluid.
Throughout this description, the terms xe2x80x9cstandard protocolsxe2x80x9d and xe2x80x9cstandard procedures,xe2x80x9d when used in the context of molecular cloning techniques, are to be understood as protocols and procedures found in an ordinary laboratory manual such as Current Protocols in Molecular Biology, editors F. Ausubel et al., John Wiley and Sons, Inc. 1994, or Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A laboratory manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 1989.